CN106188151B - A kind of ionic phosphorescent iridium complex probe based on pyridiniujm and preparation method thereof and biologic applications - Google Patents
A kind of ionic phosphorescent iridium complex probe based on pyridiniujm and preparation method thereof and biologic applications Download PDFInfo
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- CN106188151B CN106188151B CN201610528080.7A CN201610528080A CN106188151B CN 106188151 B CN106188151 B CN 106188151B CN 201610528080 A CN201610528080 A CN 201610528080A CN 106188151 B CN106188151 B CN 106188151B
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- 239000000523 sample Substances 0.000 title claims abstract description 24
- 229910052741 iridium Inorganic materials 0.000 title claims abstract description 23
- GKOZUEZYRPOHIO-UHFFFAOYSA-N iridium atom Chemical compound [Ir] GKOZUEZYRPOHIO-UHFFFAOYSA-N 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 238000003384 imaging method Methods 0.000 claims abstract description 11
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims abstract description 9
- 230000001413 cellular effect Effects 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 8
- 239000003446 ligand Substances 0.000 claims abstract description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 6
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 4
- NBPGPQJFYXNFKN-UHFFFAOYSA-N 4-methyl-2-(4-methylpyridin-2-yl)pyridine Chemical group CC1=CC=NC(C=2N=CC=C(C)C=2)=C1 NBPGPQJFYXNFKN-UHFFFAOYSA-N 0.000 claims description 3
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 claims description 2
- 238000010668 complexation reaction Methods 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- 238000006198 methoxylation reaction Methods 0.000 claims description 2
- 229910006400 μ-Cl Inorganic materials 0.000 claims description 2
- HZNVUJQVZSTENZ-UHFFFAOYSA-N 2,3-dichloro-5,6-dicyano-1,4-benzoquinone Chemical compound ClC1=C(Cl)C(=O)C(C#N)=C(C#N)C1=O HZNVUJQVZSTENZ-UHFFFAOYSA-N 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 abstract description 36
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 abstract description 15
- 108010024636 Glutathione Proteins 0.000 abstract description 12
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 abstract description 9
- 235000018417 cysteine Nutrition 0.000 abstract description 9
- 238000001514 detection method Methods 0.000 abstract description 9
- 239000000463 material Substances 0.000 abstract description 4
- 239000011365 complex material Substances 0.000 abstract description 2
- 229910052751 metal Inorganic materials 0.000 abstract 2
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 abstract 1
- 125000004122 cyclic group Chemical group 0.000 abstract 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 abstract 1
- 239000002184 metal Substances 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene chloride Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 20
- 239000000243 solution Substances 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 17
- 229960003180 glutathione Drugs 0.000 description 14
- 230000003834 intracellular effect Effects 0.000 description 9
- HYHCSLBZRBJJCH-UHFFFAOYSA-M sodium hydrosulfide Chemical compound [Na+].[SH-] HYHCSLBZRBJJCH-UHFFFAOYSA-M 0.000 description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 239000007832 Na2SO4 Substances 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 238000000295 emission spectrum Methods 0.000 description 3
- -1 filters Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- VQGHOUODWALEFC-UHFFFAOYSA-N 2-phenylpyridine Chemical compound C1=CC=CC=C1C1=CC=CC=N1 VQGHOUODWALEFC-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 125000005997 bromomethyl group Chemical group 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000006392 deoxygenation reaction Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 229910052723 transition metal Inorganic materials 0.000 description 2
- 150000003624 transition metals Chemical class 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- GTOQQRQDAUQKAN-UHFFFAOYSA-N 3-(bromomethyl)-2-pyridin-2-ylpyridine Chemical group BrCC1=CC=CN=C1C1=CC=CC=N1 GTOQQRQDAUQKAN-UHFFFAOYSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical class [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 1
- 229910021135 KPF6 Inorganic materials 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- WEVYAHXRMPXWCK-FIBGUPNXSA-N acetonitrile-d3 Chemical compound [2H]C([2H])([2H])C#N WEVYAHXRMPXWCK-FIBGUPNXSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000571 coke Substances 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000024531 detection of redox state Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 244000144992 flock Species 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- JVZRCNQLWOELDU-UHFFFAOYSA-N gamma-Phenylpyridine Natural products C1=CC=CC=C1C1=CC=NC=C1 JVZRCNQLWOELDU-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000010335 redox stress Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- PNGLEYLFMHGIQO-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methoxyanilino)-2-hydroxypropane-1-sulfonate;dihydrate Chemical compound O.O.[Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(OC)=C1 PNGLEYLFMHGIQO-UHFFFAOYSA-M 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0033—Iridium compounds
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/18—Metal complexes
- C09K2211/185—Metal complexes of the platinum group, i.e. Os, Ir, Pt, Ru, Rh or Pd
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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Materials Engineering (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Ionic phosphorescent iridium complex probe that the present invention relates to a kind of based on pyridiniujm and preparation method thereof and biologic applications, more particularly to a kind of phosphorescent iridium complex for detecting the reducing substances with sulfydryl such as reduced glutathione, cysteine, hydrogen sulfide and its detection cellular redox state application, belong to organic photoelectric functional material technical field.Such complex material is shown below by cyclic metal complexes, metal center (iridium) and the assistant ligand composition for being modified with pyridinium group, structure.Complex described in the invention has broad application prospects in terms of detection cellular redox state, phosphorescent lifetime imaging and TIME RESOLVED TECHNIQUE.
Description
Technical field
Ionic phosphorescent iridium complex probe that the present invention relates to a kind of based on pyridiniujm and preparation method thereof and biology are answered
With, and in particular to a kind of preparation method of the phosphorescent iridium complex material based on pyridiniujm and its in the cell redox state are examined
It surveys, the application in terms of phosphorescent lifetime imaging and TIME RESOLVED TECHNIQUE, belongs to organic photoelectric functional material technical field.
Background technology
Redox state is a term for being widely used in the fields such as free radical and oxidative stress.Intracellular oxidation is also
Former environment is determined by intracellular redox stress.It is to be in normal intracellular, intracellular oxidation pressure and reduction pressure
Balance, oxidation pressure is mainly determined by the content of intracellular reactive oxygen molecule and restores pressure mainly by some sulfydryl albumen and reduction
Property enzyme and amino acid determine.Intracellular redox state once due to oxidative stress disequilibrium, can to protein,
The structure and function of DNA and lipid causes irreversible destruction.Therefore, the balance of cellular redox state is to the body that sustains life
Normal physiological activity it is most important, therefore exploitation can be used for monitor cellular redox state probe be of great significance.
Compared to fluorescence probe, the phosphorescent iridium complex based on triplet state transition transmitting has relatively long luminescent lifetime,
Make a kind of ideal luminescence probe that can be used for bio-imaging label.In recent years, matched with phosphorescence transition metal iridium
It is the great interest that Imaging-PAM dye molecule causes people to close object, this is because phosphorescence transition metal complex of iridium has
There are following several big features：With larger quantum efficiency, larger Stokes displacements and longer emission lifetime.It is shone using it
The characteristics of long lifespan, can be such that phosphorescent signal is distinguished with the background fluorescence signal in organism by using TIME RESOLVED TECHNIQUE,
Other interference are avoided, and superior photostability is convenient for observation for a long time.Therefore, exploitation, which has, can be used for living body detection
Long lifetime phosphorescent probe is of great significance.
Invention content
In order to solve the above technical problems, the purpose of the present invention is to provide a kind of, the ionic phosphorescent iridium based on pyridiniujm is matched
Physical prospecting needle is closed, their preparation method is provided, and proposes this kind of complex in the detection of redox state in the cell, biomarker
With the application in cell imaging field.
In order to solve the above-mentioned technical problem, technical solution proposed by the present invention is：A kind of ionic phosphorus based on pyridiniujm
Light complex of iridium probe contains pyridiniujm on assistant ligand, which has the following structure：
Another technical solution proposed by the present invention is：A kind of ionic phosphorescent iridium complex probe preparation based on pyridiniujm
The synthetic route of method is as follows：
Specifically 4,4 '-dimethyl -2,2 '-bipyridyl is through peroxidating, methoxylation, the reduction of ester, hydrobromic acid substitution four
N^N assistant ligands are obtained by the reaction in step, are then prepared by complexation reaction with iridium dichloro bridge ((C^N) 2Ir (μ-Cl) 2Ir (C^N) 2)
Complex intermediate is obtained, is then 1 by intermediate and pyridine equivalent proportion:2 are added excessive hexafluorophosphoric acid in acetonitrile after dissolving
Potassium is stirred overnight to obtain title complex.
Another technical solution proposed by the present invention is：The ionic phosphorescent iridium complex is applied to cellular redox state
Detection.
Preferably, which is applied to phosphorescent lifetime imaging and TIME RESOLVED TECHNIQUE.
Advantageous effect：
Contain pyridiniujm on its C^N ligand of the invention, can be used to detect cellular redox state.
The material that the present invention synthesizes is used as cellular oxidation ortho states and detects phosphorescence probe, in reduced glutathione cysteine
Simultaneously blue shift, detection result are notable for phosphorescent emissions enhancing in the presence of equal biological micromolecules.
This probe material has low bio-toxicity, and is easily accessible in cell cytosol, and the long phosphorescent emissions service life can be used
TIME RESOLVED TECHNIQUE is mutually distinguished with background fluorescence signal to reduce signal-to-noise ratio so that this kind of probe can be imaged by the service life and the time
The detection of cellular redox state is realized in gate technique imaging, improves accuracy of detection.
Description of the drawings
The present invention is described further below in conjunction with the accompanying drawings.
Fig. 1 are the hydrogen nuclear magnetic resonance spectrogram of complex Ir1 in embodiment 2；
Fig. 2 are the carbon-13 nmr spectra figure of complex Ir1 in embodiment 2；
Fig. 3 are phosphorescence emission spectras of the phosphorescence probe Ir1 in PBS buffer solutions in embodiment 3 to reduced form gluathione
The response of peptide (GSH), cysteine (Cys), NaHS；
Fig. 4 be embodiment 4 in service life of the phosphorescence probe Ir1 in PBS buffer solutions to reduced glutathione (GSH),
The response of cysteine (Cys), NaHS；
Fig. 5 are that phosphorescence probe Ir1 is applied to the copolymerization coke of hydrogen reduction state detection and fluorescence longevity in living cells in embodiment 5
Order images.
Specific implementation mode
The content of patent for a better understanding of the present invention is further illustrated the present invention below by specific example
Technical solution.But these embodiments are not intended to limit the present invention.
Embodiment 1
Complex intermediate [Ir (pq)2-bpy-Br]+PF6 -Synthesis
The synthesis of (1) 4,4 '-two bromomethyl -2,2 '-bipyridyl and pyridiniujm ligand
I) take 4,4 '-dimethyl -2,2 '-bipyridyl 5g that the 250mL single port bottles with stirrer are added, are added 80mL98%'s
Dense H2SO4It is placed under water bath condition and is vigorously stirred, and slowly (ensure that temperature will not be ramping up) and 17g K are added2Cr2O7, then
65 DEG C of stirring 6h are placed it in, is cooled to room temperature, water is added to filter, dry white powder.
II) I products therefrom 2g and 10mL 98%H are taken2SO4, 100mL CH3OH is added in the single port bottle with stirrer, and 105
It DEG C is refluxed overnight, reaction terminates to add in a large amount of water white flock precipitate occurs, is slowly added to NaOH solution tune pH about 9.0, uses
CH2Cl2Extraction retains organic phase, anhydrous Na2SO4After drying, solvent evaporated obtains white crystal, yield 88%.
III) II products therefrom 630mg and NaBH is taken41g is added in the 250mL single port bottles with stirrer, and it is tight that 80mL is added
Lattice remove water the ethanol solution of deoxygenation, 80 DEG C of reflux 3h, and reaction terminates addition 20mL and is saturated NH4Cl solution, is evaporated ethyl alcohol, uses acetic acid
Ethyl ester is extracted with water, is retained ethyl acetate layer, is added anhydrous Na2SO4Dry, solvent evaporated, vacuum drying obtains white powder
End.
IV) gained intermediate product 100mg, 6mL 48%HBr solution adds to the single port bottle with stirrer in taking III, slowly adds
Enter the dense H of 9mL2SO4, dissolution of raw material is placed in 100 DEG C of oil bath agitated kettles the 6h that flows back, and reaction terminates, and a small amount of water is added, while stirring
Add KOH tune pH to 7.0, generate white precipitate, filters, CH2Cl2Lysate uses anhydrous Na2SO4It is dry, it is evaporated after filtering molten
Agent, vacuum drying obtain 4,4 '-two bromomethyl -2,2 '-bipyridyls, yield 45%.
(2) complex intermediate [Ir (pq)2-bpy-Br]+PF6 -Synthesis
Phenylpyridine is taken to close 4,4 '-two bromomethyl -2 iridium chlorine bridge dimer 200mg, 140mg, 2 '-bipyridyls, hexafluoro phosphorus
Sour potassium (KPF6) 300mg, dichloromethane 20mL, absolute methanol 10mL sequentially add in the two mouth flask with stirrer, and nitrogen is protected
Shield, return stirring for 24 hours, with dichloromethane dilute reaction solution, filter to obtain orange red dichloromethane clarified solution, after solvent evaporated, use
Dichloromethane/ethyl acetate (V:V=20:1) it is complex intermediate, yield 86% that column chromatography, which obtains orange powder,.
1H NMR(400MHz,CDCl3)δ(ppm):8.65(m；2H)；7.91-7.88(m；4H)；7.76(t；J=
8.00Hz；2H)；7.68(d；J=7.60Hz；2H)；7.53-7.51(m；3H)；7.46(dd；J1=1.20Hz；J2=5.60Hz；
1H)；7.09-7.02(m；4H)；6.92(t；J=7.20Hz；2H)；6.27(d；J=7.60Hz；2H)；4.80(s；2H)；4.63
(s；2H).
Embodiment 2
Title complex Ir1 [Ir (pq)2-bpy-py]3+3PF6 -Synthesis
Weigh complex intermediate [Ir (pq)2-bpy-Br]+PF6 -(1mmol), pyridine (2mmol) and excessive hexafluorophosphoric acid
Potassium powder is added in the two-neck bottle with stirrer, and nitrogen-is vacuumized-protected on biexhaust pipe and is vacuumized, is moved in circles three times, finally
Using the entire reaction system of nitrogen protection.Injection 5mL strictly removes water the acetonitrile solution of deoxygenation, and 80 DEG C are stirred at reflux 10h.Reaction knot
Shu Hou is cooled to room temperature, and solvent evaporated obtains crude product with column chromatography, and solvent is dichloromethane and methanol (v:V=10:
1) it is title complex Ir1 that crude product acetonitrile and recrystallize with dichloromethane, which, are obtained red crystals product,.
1H NMR(400MHz,CD3CN-d3) δ=8.72-8.55 (m, 6H)；8.34(dd,J1=20.8, J2=8.8Hz,
4H)；8.23 (d, J=5.8Hz, 2H)；8.16-8.05(m,6H)；8.02(s,2H)；7.83 (d, J=7.1Hz, 2H)；7.46-
7.32(m,4H)；7.22-7.11(m,4H)；6.98-7.03(m,2H)；6.85-6.75(m,2H)；6.47 (d, J=7.4Hz,
2H)；5.75(s,4H).
13C NMR (100MHz, CD3CN, 298K) δ=171.23,156.80,151.12,150.32,148.48,
148.42,147.09,146.38,146.22,141.50,135.39,132.36,131.88,130.51,130.37,129.19,
128.86,128.76,128.06,125.67,124.70,124.42,119.32
Nuclear magnetic resonance spectroscopy, carbon spectrum are as shown in Figure 1 and Figure 2.
Embodiment 3
Sound of the emission spectrum of phosphorescence probe Ir1 to reductive glutathione (GSH), cysteine (Cys), NaHS
Ying Xing
Complex Ir1 is made into 1.0 × 10-3It is molten to 990 μ L PBS bufferings to pipette 10 μ L solution for the acetonitrile solution of mol/L
In liquid, make its concentration dilution to 1.0 × 10-5Mol/L is tested, be separately added into 50 times of equivalents reduced glutathione, half
Cystine, sodium hydrosulfide aqueous solution, heating stirring is centrifuged in 10000 rotating speeds after five minutes in 37 DEG C of waters bath with thermostatic control, makes reduced form
Glutathione etc. is fully reacted with complex Ir1, then takes supernatant liquor to test its fluorescence emission spectrum, as shown in Figure 2.Cooperation
Object Ir1 solution maximum emission peak is in 635nm or so, after reduced glutathione, cysteine and NaHS is added, solution
Luminous intensity is remarkably reinforced and maximum emission peak blue shift, wherein luminous intensity variations after cysteine and NaHS are added
It is apparent that reduced glutathione is not added.Complex Ir1 and reduced glutathione, the half Guang ammonia that 50 times of equivalents are added
Emission spectrum after acid, sodium hydrosulfide aqueous solution is as shown in Figure 3.
Embodiment 4
Response of the service life of phosphorescence probe Ir1 to reductive glutathione (GSH), cysteine (Cys), NaHS
Complex Ir1 is made into 1.0 × 10-3It is molten to 990 μ L PBS bufferings to pipette 10 μ L solution for the acetonitrile solution of mol/L
In liquid, make its concentration dilution to 1.0 × 10-5Mol/L is tested, and is tested the service life of complex Ir1 and is buffered to the PBS of Ir1
The reductive glutathione (GSH), cysteine (Cys), sodium hydrosulfide aqueous solution of 100 times of equivalents are added in liquid, in 37 DEG C of perseverances
Heating stirring is centrifuged in 10000 rotating speeds after five minutes in tepidarium, and supernatant liquor is taken to carry out life test, complex and addition
Lifetime change after reducing agent is as shown in Figure 4.
Embodiment 5：Phosphorescence probe Ir1 is applied to living cells imaging experiment
Hela cells are cultivated according to American Type Tissue Culture Collection regulations.Hela
Cell is incubated 30 minutes with 10 μM of complex Ir1 solution at 37 DEG C, is washed 3 times with culture solution, the Hela of complex Ir1 labels
Cell uses sulphydryl activity inhibitor N-ethylomaleimide (NEM) and reduced glutathione (GSH) solution to exist respectively
It is respectively incubated under the conditions of 37 DEG C 30 minutes, is washed 3 times with culture medium later, is focused into altogether with 405nm wavelength activated cell
Picture and phosphorescent lifetime imaging, co-focusing imaging, which is shown, is evident that phosphorescence in living cells endochylema, and the Hela being incubated with GSH
Phosphorescence intensity in the cell that phosphorescence intensity ratio NEM in cell is incubated is high, and the thin of NEM incubations is added in phosphorescent lifetime imaging display
Intracellular average life span, which is less than, is added the cell that GSH is incubated, and the content of intracellular GSH determines intracellular reduction-state, is added
The cell reduction-state that GSH is incubated, which is higher than, is added the cell that NEM is incubated, as shown in figure 5, this is the result shows that phosphorescence probe Ir1 tools
There is preferable Cell permeable, be mainly distributed on cytosolic domain, can be used for the detection of living cells internal oxidition reduction-state.
Claims (4)
1. a kind of ionic phosphorescent iridium complex probe based on pyridiniujm, it is characterised in that contain pyridine on its assistant ligand
Salt, the complex of iridium have the following structure：
2. a kind of preparation method of the ionic phosphorescent iridium complex probe based on pyridiniujm as described in claim 1, special
Sign is that the synthetic route of the preparation method is as follows：
Specifically 4,4 '-dimethyl -2,2 '-bipyridyl replaces four steps anti-through peroxidating, methoxylation, the reduction of ester, hydrobromic acid
Should obtain N^N assistant ligands, then with iridium dichloro bridge ((C^N)2Ir(μ-Cl)2Ir(C^N)2) be prepared by complexation reaction
Then intermediate and pyridine equivalent proportion are 1 by complex intermediate:2 are added excessive Potassium Hexafluorophosphate in acetonitrile after dissolving stirs
It mixes and obtains title complex overnight.
3. a kind of biologic applications of the ionic phosphorescent iridium complex probe based on pyridiniujm as described in claim 1, special
Sign is that the ionic phosphorescent iridium complex is detected applied to cellular redox state.
4. a kind of biologic applications of the ionic phosphorescent iridium complex probe based on pyridiniujm as described in claim 1, special
Sign is that the ionic phosphorescent iridium complex is applied to phosphorescent lifetime imaging and TIME RESOLVED TECHNIQUE.
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| EP0602488A1 (en) * | 1992-12-15 | 1994-06-22 | Asulab S.A. | Complexes of a transition metal with 2,2-bipyridine ligands substituted by at least one ammonium alkyl radical, process for their fabrication and their use as redox mediator |
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| EP0602488A1 (en) * | 1992-12-15 | 1994-06-22 | Asulab S.A. | Complexes of a transition metal with 2,2-bipyridine ligands substituted by at least one ammonium alkyl radical, process for their fabrication and their use as redox mediator |
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| Tuning the charge-separated lifetimes of ruthenium(Ⅱ) polypyridyl-viologen dyads and ruthenium(Ⅱ) polypyridyl-viologen triads by the formation of supramolecular assemblies with crown ethers;Volker Schild等;《The Journal of Physical Chemistry A》;20020918;第106卷(第40期);第9149-9158页 * |
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